The invention relates to methods for making hybridomas which produce monoclonal immunoglobulin. More particularly, the invention relates to methods for making polyomas which express monoclonal bi-specific immunoglobulin.
There are five classes of naturally occurring immunoglobulins, viz. IgA, IgD, IgE, IgG, and IgM. IgG is the most common immunoglobulin. Immunoglobulins include at least two peptide chains, viz. a heavy chain and a light chain. Each light chain is linked by disulfide bonds to a corresponding heavy chain. Bi-valent immunoglobulin includes two pairs of paired heavy and light chains. Each of the two heavy chains are linked to one another by disulfide bonds. Each class of immunoglobulin is characterized by the structure of its heavy chain. In turn, each class of immunoglobulin may be subdivided into subclasses which are characterized by more subtle distinctions within the structure of the heavy chains. And finally, immunoglobulins can be further distinguished by the structure of the light chain, viz. either lambda or kappa light chains. Either type of light chain may occur in combination with any of the classes or subclasses of heavy chains.
Each of the heavy and light chains are divided into constant regions and variable regions. The constant regions provide the over all molecular structure of each class of immunoglobulin and the site or focus of various molecular interactions with respect to regulatory factors. The variable regions of paired heavy and light chains are juxtaposed to one another to form a combined variable region. Each combined variable region defines an antigen binding site and a framework for the antigen binding site. An antigen binding site is a site on the immunoglobulin to which the antigen binds. Each antigen binding site corresponds to one valency. Bi-valent immunoglobulins include two antigen binding sites. The antigen binding sites of naturally occurring bi-valent immunoglobulin are identical to one another, i.e. naturally occurring immunoglobulins are mono-specific.
Immunoglobulins are produced by antibody expressing cells, e.g. plasma cells and various other activated B lymphocytes. Any given naturally occurring antibody expressing cell expresses only one type of mono-specific immunoglobulin. However, the vascular and lymphatic systems contain many different such antibody expressing cells. Hence, serum antibody may include many different immunoglobulins having many different specificities. Serum antibody is therefore said to be polyclonal.
In 1975, Kohler and Milstein (Nature, vol. 256, pp 495-497) developed a technique for making hybridomas. Hybridomas are hybrid cells created by fusing lymphocytes or other antibody expressing cells with myeloma cells, i.e. fusion partner cells. Myeloma cells are neoplastic plasma cells which preferably do not express antibody and which which are culturable, i.e. they may be maintained in culture indefinitely. On the other hand, lymphocytes are antibody expressing leukocytes. Lymphocytes are nonculturable, i.e. they can not be maintained in culture for an extended period. Some of the cellular fusion products of these two cell types will combine the properties of both parent cells, viz. the ability to express immunoglobulin and the ability to be maintained in culture for an extended period. The specificity of the immunoglobulin expressed by the cellular fusion product or hybridoma is determined by the specificity of the immunoglobulin expressed by the parent lymphocyte. When properly screened and cloned, the hybridomas can be grown in culture for an extended period and will produce large quantities of uniform mono-specific immunoglobulin, i.e. monoclonal immunoglobulin.
The advent of hybridoma technology caused a rapid proliferation of new developments and applications for monoclonal immunoglobulin. Included within the new developments were new methods for fabricating bi-specific immunoglobulin.
A bi-specific immunoglobulin is an immunoglobulin having two different antigenic specificities. Bi-specific immunoglobulin is sometimes known as bi-functional immunoglobulin. Bi-specific immunoglobulin is useful in various immunoassay procedures, therapies, and other uses.
The initial efforts to produce bi-specific immunoglobulin employed chemical methods on polyclonal immunoglobulin. The first step of these methods involved a reductive dissociation of each of two samples of polyclonal immnoglobulin. The two samples of polyclonal immunglobulin are chosen to have different specificities from one another. After the reductive dissociation, the two samples are then combined and allowed to undergo an oxidative re-association. In the first step, the disulfide linkages of the polyclonal immunoglobulin are broken by exposure to reducing conditions. In the second step, the dissociated subunits are then allowed to reassemble under oxidating conditions. It was found that the reassembly process is somewhat random and that the reassembly of immunoglobulin into the desired bi-specific form was somewhat inefficient.
A more efficient method for chemically producing bi-specific immunoglobulin was recently disclosed by Maureen Brennan et al. (Science, vol. 229, pp 81-83 (1985)). Brennan discloses the use of monoclonal immunoglobulin as the starting material. Two monoclonal antibodies, each having a different antigenic specificity, are employed. Unfortunately, the method of Brennan involves a limited pepsin hydrolysis of the starting antibodies and therefore produces a bi-specific F(ab')2 fragment instead of an intact immunoglobulin molecule (IgG). However, the chemistry of Brennan's process is relatively efficient.
In 1984, Christopher Reading (U.S. Pat. No. 4,474,893) disclosed a method for making triomas and quadromas which express recombinant bi-specific immunoglobulin. Reading defines a trioma to be a cellular fusion product which results from the fusion of a lymphocyte and a hybridoma cell or other such fusion partner cells which are culturable and antibody expressing. A quadroma is defined to be the cellular fusion product which results from the fusion of two hybridomas, i.e. two fusion partner cells. Quadromas are sometimes also known as hybrid-hybridomas. With respect to the trioma, Reading points out that, if the hybridoma and the lymphocyte each express different antibodies, i e. immunoglobulin having different specificities, then the resultant trioma may express a recombinant bi-specific immunoglobulin. Similarly, with respect to the quadroma, if the two hybridomas each express different antibodies, then the resultant quadroma may express a recombinant bi-specific immunoglobulin. It is assumed that, for any given trioma or quadroma, the sites of recombination will be randomly distributed within the genome.
Other workers have made hetero-hybridomas. A hetero-hybridoma is a cellular fusion product derived from the cells of two different species. The fusion may be between a myeloma from one species and a lymphocyte from a different species; between a hybridoma derived from one species and a lymphocyte from a different species; or between two hybridomas derived from different species. These hetero-hybridomas express recombinant monoclonal immunoglobulin which includes portions of each of the two species. However, such hetero-hybridomas have not been reported to produce bi-specific monoclonal immunoglobulin.
The relative efficiency of the use of triomas and quadromas for producing bi-specific immunoglobulin is compared to the chemical methods by Brennan (supra). Brennan states that, employing her method and starting with a supply of two types of monoclonal immunoglobulin, a chemist can produce pure bi-specific immunoglobulin within one week. By comparison, Brennan indicates that it would require a significantly longer period of time to produce purified bi-specific immunoglobulin if one were required to produce a trioma or quadroma ab initio. Hence, one can infer from Brennan that the speed with which bi-specific immunoglobulin can be made is an important factor for many users.
Indeed, in certain clinical situtations, it may be very important to produce bi-specific monoclonal antibodies with great speed. Reading indicates that bi-specific monoclonal antibodies can be employed as reagents for the treatment of tumors. One valency of the reagent has a specificity for the tumor; the other valency of the reagent has a specificity for a drug or toxin which is to be targeted against the tumor. However, if a patient develops a tumor having an unusual or unique tumor specific antigen, it may be necessary to make a new bi-specific monoclonal immunoglobulin to correspond to such tumor specific antigen. If a reagent employing a bi-specific monoclonal immunoglobulin is to have clinical utility, the patient's physician must have quick access to the reagent. If the reagent is custom made for the patient, it should be capable of being produced quickly.
Brennan points out that chemical method for making bi-specific immunoglobulin can be quicker than cellular methods. However, Brennan's method is premised on the pre-existance of a supply of the appropriate monoclonal immunoglobulin. If a bi-functional monoclonal immunoglobulin needs to be custom taylored to the antigenic specificity of a patient's tumor, the difference in the speed between Brennan's method and Reading's method is greatly reduced.
What is needed is a fast track method for producing bi-specific monoclonal immunoglobulin. Unlike prior art chemical methods, the fast track method should be adaptable to the production of bi-specific monoclonal immunoglobulin with new antigenic specificities. What is needed is a fast track method for producing bi-specific monoclonal immunoglobulin which by-passes the serial fusions and clonings which are employed in prior art cellular methods for making triomas and quadromas.